The immobilization of deoxyribonucleic acid (DNA) onto support surfaces has become an important aspect in the development of DNA-based assay systems, including the development of microfabricated arrays for DNA analysis. See, for instance, “The Development of Microfabricated Arrays of DNA Sequencing and Analysis”, O'Donnell-Maloney et al., TIBTECH 14:401-407 (1996). Generally, such procedures are carried out on the surface of microwell plates, tubes, beads, microscope slides, silicon wafers or membranes.
A commonly used method for immobilizing cDNA's or PCR products into arrays is to first coat glass slides with polylysine, then apply the DNA and illuminate with UV light to photocrosslink the DNA onto the polylysine (for example, see Schena M, Shalon D, Heller R, Chai A, Brown P O, Davis R W, “Parallel Human Genome Analysis: Microarray-based Expression Monitoring of 1000 Genes”, Proc. Natl. Acad. Sci. USA 93(20):10614-9 (1996)). One disadvantage of this approach is that the UV crosslinking causes undesirable damage to the DNA that is not all useful for the immobilization. Another disadvantages of this approach is that UV crosslinking tends to be limited to longer nucleic acids (e.g., over about 100-mers), as provided by cDNA's and PCR products (and in contrast to the shorter nucleic acids typically formed by synthesis and referred to as “oligonucleotides”). It appears that the potential damage induced by UV radiation (e.g., the formation of thymine dimers) is simply too great, and/or the extent of immobilization is insufficient, to permit shorter nucleic acids to be used. A population of longer nucleic acids, however, even when crosslinked by UV, will typically provide ample undamaged regions sufficient to permit accurate hybridization.
Only relatively few approaches to immobilizing DNA, to date, have found their way into commercial products. One such product for immobilizing oligonucleotides onto microwell plates is known as [“NucleoLink®”] NUCLEOL™, and is available from Nalge Nunc International (see, e.g., Nunc Tech Note Vol. 3, No. 17). In this product, the DNA is reacted with a carbodiimide to activate 5′-phosphate groups, which then react with functional groups on the surface. Disadvantages of this approach are that it requires the extra step of adding the carbodiimide reagent as well as a five hour reaction time for immobilization of DNA, and it is limited to a single type of substrate material.
As another example, Pierce has introduced a proprietary DNA immobilization product known as [“Reacti-Bind™] REACTI-BINDTM DNA Coating Solutions” (see “Instructions REACTI-BINDTM DNA Coating Solution” January, 1997). This product is a solution that is mixed with DNA and applied to surfaces such as polystyrene or polypropylene. After overnight incubation, the solution is removed, the surface washed with buffer and dried, after which it is ready for hybridization. Although the product literature describes it as being useful for all common plastic surfaces used in the laboratory, it does have some limitations. For example, Applicants were not able to demonstrate useful immobilization of DNA onto polypropylene using the manufacturer's instructions. Furthermore, this product requires large amounts of DNA. The instructions indicate that the DNA should be used at a concentration between 0.5 and 5 μg/ml.
Corning sells a product called “DNA-BIND™” for use in attaching DNA to the surface of a well in a microwell plate (see, e.g., the DNA-BIND™ “Application Guide”). The surface of the DNA-BIND™ plate is coated with an uncharged, nonpolymeric, low molecular weight, heterobifunctional reagent containing an N-oxysuccinimide (NOS) reactive group. This group reacts with nucleophiles such as primary amines. The heterobifunctional coating reagent also contains a photochemical group and spacer arm which covalently links the reactive group to the surface of the polystyrene plate. Thereafter, amine-modified DNA can be covalently coupled to the NOS surface. The DNA is modified by adding a primary amine either during the synthesis process to the nascent oligomer or enzymatically to the preformed sequence. Since the DNA-BIND™ product is polystyrene based, it is of limited use for those applications that require elevated temperatures such as thermal cycling. Corning also sells an aminosilane-coated glass slide, under the tradename CMT-GAPS™ coated slides, which uses the same protocol as polylysine-coated slides for immobilizing DNA into microarrays.
TeleChem International, Inc. sells slides coated with an aldehyde silane as well as an aminosilane-coated slide. The aldehyde silane slides have very high backgrounds when fluorescence is used for detection. They also require an additional reduction step for stable immobilization.
Finally, SurModics, Inc., the assignee of the present invention, has recently introduced a coated glass slide, under the tradename 3D-Link™ that consists of a hydrophilic polymer containing amine-reactive ester groups immobilized onto the surface. For best results, this product also requires amine modification of the DNA to be immobilized. As expected, however, the reactive ester groups tend to be hydrolytically unstable, which limits the amount of time arrays can be printed without some loss of performance to approximately eight hours.
The role of epoxide groups, in the course of binding or immobilizing nucleic acids, has been described in various ways as well. For instance, Shi et al., U.S. Pat. No. 5,919,626 describe the attachment of unmodified nucleic acids to silanized solid phase surfaces. The method involves the use of conventional nonpolymeric reagents such as mercapto-silanes and epoxy-silanes which bond to the surface by forming siloxane bonds with OH groups on the glass surface.
See also, U.S. Pat. No. 5,925,552 (Keogh, et al., “Method for Attachment of Biomolecules to Medical Devices Surfaces”), which provides a method for forming a coating of an immobilized biomolecule on a surface of a medical device to impart improved biocompatibility for contacting tissue and bodily fluids. One such method includes converting a biomolecule comprising an unsubstituted amide moiety into an amine-functional material, combining the amine-functional material with a medical device biomaterial surface comprising a chemical moiety (such as, for example, an aldehyde moiety, an epoxide moiety, an isocyanate moiety, a phosphate moiety, a sulphate moiety or a carboxylate moiety) which is capable of forming a chemical bond with the amine-functional material, to bond the two materials together to form an immobilized biomolecule on a medical device biomaterial surface. Included within the long list of biomolecules described as being useful in this patent were “a DNA segment, a RNA segment, a nucleic acid” and others.
Also on the subject of epoxides, Nagasawa et al., (J. Appl. Biochem. 7:430-437, 1985) describe the use of Sepharoses activated with epichlorohydrin or bisoxirane (both of which provide epoxide groups) for immobilizing DNA as immunosorbents for DNA antibodies. See also, Wheatley, et al., J. Chromatog. A 726:77-90 (1996) and Potuzak, et al., Nucl. Acids Res. 5:297-303 (1978).
To date, however, there appears to be no description in the art, let alone commercial products, that provide an optimal combination of such properties as hydrolytic stability, ease of use, minimized DNA damage (due to exposure to crosslinking radiation), and the ability to immobilize underivatized nucleic acids and/or shorter nucleic acid segments. In turn, there appear to be no products presently available, nor descriptions in the art, that provide or suggest the ability to use polymer-pendent epoxide groups adapted to immobilize either short or long nucleic acids, let alone in both derivatized and underivatized forms, and suitable for immobilization onto surfaces.
Finally, Surmodics, Inc., the assignee of the present invention, has previously described a variety of applications for the use of photochemistry, and in particular, photoreactive groups, e.g., for attaching polymers and other molecules to support surfaces. See, for instance, U.S. Pat. Nos. 4,722,906, 4,979,959, 5,217,492, 5,512,329, 5,563,056, 5,637,460, 5,714,360, 5,741,551, 5,744,515, 5,783,502, 5,858,653, and 5,942,555.